Critique of Rogers’ so-called vanillin clock for dating the Shroud: why was Stanley T. Kosiewicz not a co-author (and where’s the data)?

STURP’s Raymond N.Rogers very last peer-reviewed paper (see title page below)  was published in 2005,  sadly the same year that he finally succumbed to a long illness.  It was the subject of a news item by the BBC. bbc rogers Some, myself  included, say it should never have been published. Unsentimental, certainly, but science has to be ruthlessly unsentimental, or it would quickly cease to be science.

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The same standards should apply to any paper submitted for publication, regardless of what the journal’s referees and editors might know about the paper’s authors, which in the case of Thermochimica Acta would have been a great deal (since  according to his wiki entry Rogers was both a founder and long-term editor of the journal). There are two chief reasons for feeling that the now widely cited paper should have been rejected. The first, covered in this posting, was the behind-the-scenes  involvement of one Dr. Stanley T.Kosiewicz in the crucial matter of Rogers’ chemical clock for dating old linen – one that relies on loss of vanillin from lignin (the latter a relatively minor component of linen threads). First, let’s see what Ray Rogers had to say concerning Stanley Kosiewicz.

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Answer: virtually nothing, just a name and location, with nothing in the references either:

No mention of Stanley T.Kosiewicz

No mention of Stanley T.Kosiewicz

Yet when you read on, it was this same Stanley T.Kosiewicz who did all the groundwork for using degraded lignin as a chemical clock. So who is he, what was his expertise in lignin, and where else might one find his experimental data, given it’s nowhere to be found in Rogers’ paper? cropped stanley t kosiewiczSo, Dr.Kosiewicz was someone who worked at the same research institute as Mr.Rogers, but there is nothing I can find on the internet (Google Scholar etc) to show that his research speciality was lignin or plant science in general. Indeed, as the above screen grab shows, he is someone who has made his name in the disposal of radioactive wastes.

This is bizarre – totally bizarre. Stanley T.Kosiewicz should have been a co-author on the paper, given the novel methodology was developed by him, and the experimental data should have been displayed, especially that which attempts to use elevated temperature as an accelerated model for ageing effects. Yes. that’s the crucial assumption made – that while the rate of lignin degradation is too slow to be detected experimentally at normal environmental temperatures, you can measure it at high temperatures – 8o, 100 degrees C etc , and calculate via back-extrapolation what it would be at normal temperatures, making “reasonable” assumptions about time-temperature relationships, i.e. kinetic modelling.

Well, I need hardly say that anyone introducing a new clock that does not read out time directly, that needs some kind of embedded software that calculates a figure, needs to be scrutinized very closely before being  accepted as valid. But that data were not provided in the Rogers’ paper, and do not even appear to be published elsewhere in the literature. And STK was NOT a “lignin man” either.

Conclusion: on this basis alone – the failure to present the data for the model employed – the paper should have been rejected outright.

However, if some think I am being too harsh in my judgement, and consider that Rogers should have been taken on trust, especially given his fast-deteriorating health, or merely asked to provide a separate co-authored paper with Kosiewicz,  let me say straightaway that there are additional grounds for thinking that the vanillin clock is/was an exceedingly weak and probably invalid model.  There are other chemical and physical considerations  that concern this retired biomedical scientist to do with the nature of lignin breakdown at the molecular level, and to the physical nature of the vanillin breakdown product (a white solid, MPt 82-84 degrees C, and reportedly reactive towards the phloroglucinol/HCl reagent used to measure UNDEGRADED ligin).

I shall stop here for now, and add more on the end of this posting in the next day or two.

Back sooner than expected:  incessant UK rain stopped daily walk:

Time now for a critical appraisal of this passage in the Rogers’ paper:

The lignin at growth nodes on the shroud’s flax fibers  (Fig. 1) did not give the usual chemical spot test for lignin (i.e., the phloroglucinol/HCl test for vanillin). The  Holland cloth and other medieval linens gave a clear test. This suggested that the rate of loss of vanillin from lignin could offer a method for estimating the age of the shroud. The phloroglucinol–hydrochloric-acid reagent detects vanillin  (4-hydroxy-2-methoxybenzaldehyde) with good sensitivity.

In the interests of chemical veracity, I feel the need to set the record straight here, or at any rate the terminology, even if the chosen reagent was the correct one for the task (if poorly explained). Phloroglucinol/HCl has long been used as a histological stain for lignin, giving a pink or red colour. It is called the Wiesner test (often mispelled Weisner). Rogers could be excused for omitting this detail, but it’s curious that the journal referees allowed him to omit any technical detail as to how it was used, notably concentrations, time etc. More importantly, where did he come by the idea that the Wiesner test depends on the presence of vanillin in lignin? Certainly not from the literature that I have been reading these last few days, which states almost unanimously that it targets predominantly the coniferaldehyde end groups of lignin.

I have taken the schematic diagram from wiki of the structure of part of a generic lignin molecule (for which it’s not possible to write a specific structure given the variation in monolignol composition) and placed a yellow rectangle around the single occurrence there of a coniferaldehyde unit:

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However that structure is not vanillin (more correctly chemically-incorporated  vanillin). It has two more carbon atoms in the side chain than does vanillin. Here are the structures of free coniferaldehyde  (blue rectangle) and vanillin (red) from a diagram in the Biochemical Journal (for which this blogger once used to referee papers):

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A current Google search offers little comfort to those who think that it’s vanillin that is measured in the Wiesner test:

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Note especially the listings for  authoritative Pomer paper (first and second). The latter states  “it is unlikely that vanillin contributes to a great extent”. Let’s go and retrieve the full abstract of that paper. (Apols for the large font – a C&P)

Protoplasma. 2002 Oct;220(1-2):17-28.

O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction.

(Caveat: the paper is focused on the highly lignified xylem of plant conducting tissue, as distinct from the bast-type fibres of flax and linen derived therefrom):

My bolding:

“However, given the relatively low abundance of 4- O-linked vanillin in lignifying cell walls and the low extinction coefficient of its red-brown phloroglucinol adduct, it is unlikely that vanillin contributes to a great extent to the phloroglucinol-positive stain reaction. These results suggest that the phloroglucinol-HCl pink stain of lignifying xylem cell walls actually reveals the 4- O-linked hydroxycinnamyl aldehyde structures contained in lignins.”

I am still trying to track down specific information on flax bast fibre lignins (max 3% total mass, the rest being celluloses, hemicelluloses, pectins etc) and have so far drawn a blank, so I think it improbable (though not impossible) that Rogers had privileged information, certainly as he had never displayed a passion for botanical detail in his other work, thinking of that astonishing  ‘blind spot’ for the primary cell wall and hemicelluloses in the context of image-receptive layers that deserved consideration before invoking his Pliny (mis)referenced starch/saponin impurity layer.

Well, if I come across any more that says that lignin, or flax lignin in particular, contains vanillin monolignol units in their structure that are primarily responsible for the red colour with the Wiesner reagent (while other sources say that vanillin, if present, probably as a natural oxidation product, gives a weaker yellow colour) I will let you know.

For now, let’s ask why or how Rogers could have been responsible for disseminating a misleading description of what he was measuring. A quote of his elsewhere provides a clue. He refers to vanillin as conferring a characteristic aroma to old lignin, recognizing that it is a breakdown product. But he did not appear to realize that it was not due to release of a combined form of vanillin, but instead required an oxidation of a coniferylaldehyde unit with loss of 2 carbon atoms. Now one may say I’m quibbbling over terminology, being excessively purist (though Rogers was the chemist, not I, being biochemically trained).

However, there is another pitfall lurking here if Rogers thought that vanillin was a volatile material, going by the aroma from old wood.  That could conceal a major source of error if his sleeping partner, Dr. S.T.Kosiewicz of the title, was not aware of it. I refer to the fact that while lignin decay can be measured by diminishing response to the Wiesner reagent, due to oxidation of coniferylaldehyde end groups and their conversion to vanillin, that depends on the vanillin escaping, so as not to interfere with the colorimetric test, even in a small way, but enough to ‘throw’ the artificially-calculated high temperature kinetics.

Now here’s the spoiler: while the nose can readily detect vanillin at exceedingly low concentrations in air, it’s in fact a white solid with a quite high melting point (82 degrees C).  So when Kosiewicz measured remaining “vanillin” (read coniferaldehyde) at 70 degrees C, he would have been measuring some of the vanillin end product that had failed to vaporise, still being a solid with low surface area.  But when he raised the temperature to 100 degrees, the vanillin would then be liquid, with a larger surface area, larger vapour pressure, and been able to escape more easily causing less interference in the measurement of remaining “lignin”. You see, the exponential nature of the reaction rate means that any small errors at the higher temperatures is likely to translate into major errors in estimating the tiny rates of decay under normal environmental temperature.

There’s an “if” here, namely  that Kosiewicz was measuring lignin by disappearance, using the Wiesner test, rather than some more specific test. But we are not told how he did his chemistry. Thus the title (and chief complaint) of this posting. Since when has it been accepted practice to  sub-contract vital research to one’s chums, possibly/probably unqualified in the topic of interest,  whose data is withheld from publication?

Update: Saturday. Yes, it’s true what wiki says – that Rogers published the very first paper to appear in Thermochimica Acta in 1970.

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Click to enlarge

(to be continued)

Further reading: Dating the Shroud by its vanilla content

Some responses from shroudstory.com

  1. Thibault HEIMBURGER
    January 31, 2014 at 6:31 pm | #7

    Colin,

    I will not discuss now the details of your critique.
    Moreover, I agree with you that some important data (mainly the data of Stanley T.Kosiewicz) are missing in Rogers’ paper.

    Let’s go now to the basic observations.
    Even if you reject the Rogers’ lignin/vanillin-CLOCK as not proved by the data we have, a hard fact remains:
    The Shroud’s flax fibers did not give “the usual chemical spot [Wiesner] test for lignin (..)” (Rogers)
    This test was performed by Rogers on his own samples and by Adler/Heller on their samples with the same negative results.

    However, according to Rogers, the “RAES THREADS, the Holland cloth, and all other medieval linens gave the test for vanillin (…)” (Rogers’ paper, p.3).

    Let’s forget for a moment the question of the clock (dating).
    Even if you disagree with Rogers’ interpretation -the “clock”- the fact that the Raes linen fibers gave a clear Wiesner test while the Shroud fibers did not give the same test remains.

    How do, you explain that ?

    • January 31, 2014 at 7:05 pm | #8

      Hello Thibault. I have been assembling that rather difficult posting in stages, given the complexity of the subject (I’m sure I do not need to tell you that lignin is complex, and so is the chemistry of its “simple” spot tests, and more so its breakdown). You will note that I said there was more to come, and you have correctly anticipated the key question, namely, that no matter what deficiencies exist in the underlying methodology or its reporting, that need not detract from the paper’s final conclusions, namely that there is a clear difference in Shroud linen, relative to Holland cloth etc, that can be linked to the age as determined by the lignin characteristics.

      Tell me this. What is the use of a clock that is either running, or has stopped completely? That is the situation with the Shroud linen. We are asked to believe that it’s the absence of Wiesner-reactive lignin that is the reason, ie that it is incredibly aged.

      Sorry, I don’t buy that. The “vanillin clock” is so poorly documented that I decline to believe that the absence of a positive test is necessarily to do with age. It could be due to any number of factors – other than age – that affected the Shroud before 1534 (approx) when the Holland cloth was attached. to patch the burn holes. What about those references to earlier attempts to test the permanence of the Shroud image by boiling in oil etc? (Sorry, do not have links to hand) They could have oxidised the more susceptible coniferaldehyde end groups of lignin too, resulting in a negative Wiesner test.

      (Coniferaldehyde – note – with 3-carbon propanal side chain – NOT vanillin as stated by Rogers which is 1-carbon only).

      Sorry, but Rogers went looking for data to support a preconception, and filtered out anything that did not suit. That’s agenda-driven science. I’m not condemning it outright – since it keeps real scientists on their toes. I’m just fascinated by the fact that the two can somehow manage to co-exist (uneasily) and offer an alternative if dubious route by which science can make progress. But how much time is wasted, fielding bad science (and Rogers’ paper WAS bad science)?.

      • February 1, 2014 at 3:41 am | #9

        PS. Another thought struck me a few minutes ago of another factor, other than age, that could account for loss of colour with phloroglucinol in the Wiesner test. According to a literature reference I can supply, phloroglucinol reacts with the free aldehyde group of coniferyaldehyde (forming an oddball pinkish cationic species).

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        So anything that blocks that aldehyde will probably prevent the reaction. A classic way of blocking aldehyde groups is to form addition products with bisulphites (essentially dissolved sulphur dioxide gas). What’s more the removal of lignin from wood in papermaking relies on that same reaction between SO2/HSO3- and free aldehyde functions.

        I’m sure I need hardly tell you that there is more than one way in which SO2 etc could have come into contact with the Shroud in its early history. It could have been used perhaps to bleach yarn (it’s the classic method for bleaching straw hats with their high lignin content, though I’m less certain about its effects on linen). Alternatively, someone may have burned sulphur candles in the vicinity of the Shroud – known from antiquity as a means par excellence for fumigation to kill insects and their larvae etc.

        Someone here was saying just a day or two ago that Rogers’ chemical clock was just as accurate as radiocarbon dating. Bless!

    • February 1, 2014 at 4:17 am | #10

      Second afterthought. Someone decided to fumigate the reliquary (see my earlier comment). They removed the TS, then inserted a lit sulphur candle. Later the candle was removed, and the TS replaced, with its long sides folded in towards the middle before folding or rolling. Residual SO2 made better contact with the central regions of the TS than with the edges. So the reactive aldehyde groups of lignin in the initially peripheral Raes threads were better protected from the SO2 than the more central threads.

      I’m not suggesting this was the actual process that gave the difference between Raes v the rest, but it’s an indication of the uncertainties that attach to using a chemical as distinct from radioactive clock, where one is at the mercy of environmental conditions that one can only guess at, as I am guessing right now.

       

      Afterthought: added Sunday 9th Feb:

      Rogers should not have been so free-and-easy with his use of the term vanillin, which over-simplifies the challenge of producing a chemical clock. He should have called it a lignin clock, making clear that that it only a small proportion of the monolignols that are measured, notably coniferaldehyde. Instead of being content to say that lignin slowly oxidises, releasing vanillin as a product with loss of a side chain,  he should have pointed out that vanillin can potentially interfere with the lignin assay, given that it has a free aldehyde group like coniferaldehyde, capable of reacting and giving colour with the Wiesner reagent. So two things need to happen for the clock to stand a chance of working in practice – initial loss of coniferaldehyde (by oxidation) and loss of any vanillin (a solid at room temperature) by sublimation, oxidation etc.

       

       

       

About Colin Berry

Retired science bod, previous research interests: phototherapy of neonatal jaundice, membrane influences on microsomal UDP-glucuronyltransferase, defective bilirubin and xenobiotic conjugation and hepatic excretion, dietary fibre and resistant starch.
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